Pharmacological profiling of sigma 1 receptor ligands by novel receptor homomer assays.

The sigma 1 receptor (σ1R) is a structurally unique transmembrane protein that functions as a molecular chaperone in the endoplasmic reticulum (ER), and has been implicated in cancer, neuropathic pain, and psychostimulant abuse. Despite physiological and pharmacological significance, mechanistic underpinnings of structure-function relationships of σ1R are poorly understood, and molecular interactions of selective ligands with σ1R have not been elucidated. The recent crystallographic determination of σ1R as a homo-trimer provides the foundation for mechanistic elucidation at the molecular level. Here we report novel bioluminescence resonance energy transfer (BRET) assays that enable analyses of ligand-induced multimerization of σ1R and its interaction with BiP. Haloperidol, PD144418, and 4-PPBP enhanced σ1R homomer BRET signals in a dose dependent manner, suggesting their significant effects in stabilizing σ1R multimerization, whereas (+)-pentazocine and several other ligands do not. In non-denaturing gels, (+)-pentazocine significantly decreased whereas haloperidol increased the fraction of σ1R multimers, consistent with the results from the homomer BRET assay. Further, BRET assays examining heteromeric σ1R-BiP interaction revealed that (+)-pentazocine and haloperidol induced opposite trends of signals. From molecular modeling and simulations of σ1R in complex with the tested ligands, we identified initial clues that may lead to the differed responses of σ1R upon binding of structurally diverse ligands. By combining multiple in vitro pharmacological and in silico molecular biophysical methods, we propose a novel integrative approach to analyze σ1R-ligand binding and its impact on interaction of σ1R with client proteins.

Impact of capillary flow hydrodynamics on carrier-mediated transport of opioid derivatives at the blood-brain barrier, based on pH-dependent Michaelis-Menten and Crone-Renkin analyses.

Most studies of blood-brain barrier (BBB) permeability and transport are conducted at a single pH, but more detailed information can be revealed by using multiple pH values. A pH-dependent biophysical model was applied to the mechanistic analysis of published pH-dependent BBB luminal uptake data from three opioid derivatives in rat: pentazocine (Suzuki et al., 2002a, 2002b), naloxone (Suzuki et al., 2010a), and oxycodone (Okura et al., 2008). Two types of data were processed: in situ brain perfusion (ISBP) and brain uptake index (BUI). The published perfusion data were converted to apparent luminal permeability values, Papp, and analyzed by the pCEL-X program (Yusof et al., 2014), using the pH-dependent Crone-Renkin equation (pH-CRE) to determine the impact of cerebrovascular flow on the Michaelis-Menten transport parameters (Avdeef and Sun, 2011). For oxycodone, the ISBP data had been measured at pH7.4 and 8.4. The present analysis indicates a 7-fold lower value of the cerebrovascular flow velocity, Fpf, than that expected in the original study. From the pyrilamine-inhibited data, the flow-corrected passive intrinsic permeability value was determined to be P0=398×10-6cm·s-1. The uptake data indicate that the neutral form of oxycodone is affected by a transporter at pH8.4. The extent of the cation uptake was less certain from the available data. For pentazocine, the brain uptake by the BUI method had been measured at pH5.5, 6.5, and 7.4, in a concentration range 0.1-40mM. Under similar conditions, ISBP data were also available. The pH-CRE determined values of Fpf from both methods were nearly the same, and were smaller than the expected value in the original publication. The transport of the cationic pentazocine was not fully saturated at pH5.5 at 40mM. The transport of the neutral species at pH7.4 appeared to reach saturation at 40mM pentazocine concentration, but not at 12mM. In the case of naloxone, a pH-dependent Michaelis-Menten equation (pH-MME) analysis of the data indicated a smooth sigmoidal transition from a higher capacity uptake process affecting cationic naloxone (pH5.0-7.0) to a lower capacity uptake process affecting the neutral drug (pH8.0-8.5), with cross-over point near pH7.4. Evidently, measurements at multiple pH values can reveal important information about both cerebrovascular flow and BBB transport kinetics.

Characterization of ligand binding to the σ(1) receptor in a human tumor cell line (RPMI 8226) and establishment of a competitive receptor binding assay.

The standard assay for the determination of σ(1) receptor affinities of novel compounds is a competitive binding assay using [(3)H]-(+)-pentazocine as radioligand and membrane preparations from guinea pig brain. Herein, a novel competitive binding assay was developed employing the hematopoietic cell line of human multiple myeloma (RPMI 8226), which expresses a large amount of the human σ(1) receptor. Membrane fragments of RPMI 8226 cells were prepared and characterized. A Western blot analysis confirmed the high density of σ(1) receptors in this cell line. Assay conditions were carefully optimized leading to an incubation period of 120 min, an incubation temperature of 37°C, and receptor material for each well was prepared from 300,000 cells. It was shown that a large excess (10 µM) of (+)-pentazocine, haloperidol, and di-o-tolylguanidine provided the same results during determination of the nonspecific binding. Saturation experiments with the radioligand [(3)H]-(+)-pentazocine led to a K(d)-value of 36±0.3 nM and a B(max)-value of 477±7 fmol/mg protein. These data resulted in approximately 122,000 σ(1) binding sites per cell. The assay was validated by using six known σ(1) ligands and eight σ(1) ligands prepared in our lab. The K(i)-values determined with RPMI 8226-derived receptor material are in good accordance with the K(i)-values obtained with guinea pig brain membrane preparations. Compared with guinea pig brain preparations, the RPMI 8226-derived receptor material represents a better standardized receptor material with a high density of human σ(1) receptors.

Synergistic effect of isopropyl myristate and glyceryl monocaprylate on the skin permeation of pentazocine.

The aim of this investigation was to assess the applicability of lipid bilayer alteration using a combination of isopropyl myristate (IPM) and glyceryl monocaprylate (GEFA-C(8)) to the enhancement of pentazocine (PTZ) permeation through hairless mouse skin. The skin permeability of PTZ was enhanced by increasing the concentration of GEFA-C(8) up to 10% w/w in combination with IPM. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and small angle X-ray diffraction (SAXD) were carried out to analyze the effects of these enhancers on the biophysical properties of the stratum corneum (SC) of the skin, and on the permeation of PTZ. ATR-FTIR studies revealed that IPM/GEFA-C(8) induced higher CH(2) stretching frequencies of SC lipids than IPM alone. SAXD showed the disappearance of long lamellar diffraction of SC lipids with IPM/GEFA-C(8), resulting in a complete loss of order of the SC lipid bilayers. When 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI), a hydrophobic fluorescence probe, was applied in IPM alone, the amount of DiI which penetrated into the intercellular space of the SC was very low, but this was markedly increased when DiI was applied in IPM/GEFA-C(8). These results indicate that the synergistic effects of IPM and GEFA-C(8) enhance transdermal permeation of PTZ by disrupting SC lipids.

Eudragit-based transdermal delivery system of pentazocine: Physico-chemical, in vitro and in vivo evaluations.

The present study was aimed to develop a matrix-type transdermal formulation of pentazocine using mixed polymeric grades of Eudragit RL/RS. The possible interaction between drug and polymer used were characterized by FTIR, DSC and X-RD. X-RD study indicates a change of state of drug from crystalline to amorphous in the matrix films prepared. The matrix transdermal films of pentazocine were evaluated for physical parameters and in vitro dissolution characteristic using Cygnus' sandwich patch holder. Irrespective of the grades of Eudragit polymer used, the thickness and weight per patch were similar. In vitro dissolution study revealed that, with an increase in the proportion of Eudragit RS (slightly permeable) type polymer, dissolution half life (t(50%)) increases and dissolution rate constant value decreases. Selected formulations were chosen for these pharmacokinetic studies in healthy rabbits. The relevance of difference in the in vitro dissolution rate profile and pharmacokinetic parameters (C(max), t(max), AUC((s)), t(1/2,) K(el), and MRT) were evaluated statistically. In vitro dissolution profiles (DRC and t(50%)) and pharmacokinetic parameters showed a significant difference between test products (P<0.01). Quantitatively good correlation was found between the percentage of drug absorbed from the transdermal patches and AUC((s)).

Development of matrix controlled transdermal delivery systems of pentazocine: In vitro/in vivo performance.

The present study aimed to develop hydroxypropyl methylcellulose based transdermal delivery of pentazocine. In formulations containing lower proportions of polymer, the drug released followed the Higuchi kinetics while, with an increase in polymer content, it followed the zero-order release kinetics. Release exponent (n) values imply that the release of pentazocine from matrices was non-Fickian. FT-IR, DSC and XRD studies indicated no interaction between drug and polymer.The in vitro dissolution rate constant, dissolution half-life and pharmacokinetic parameters (C(max), t(max), AUC(s), t(1/2), Kel, and MRT) were evaluated statistically by two-way ANOVA. A significant difference was observed between but not within the tested products. Statistically, a good correlation was found between per cent of drug absorbed from patches vs. C(max) and AUC(s). A good correlation was also observed when per cent drug released was correlated with the blood drug concentration obtained at the same time point. The results of this study indicate that the polymeric matrix films of pentazocine hold potential for transdermal drug delivery.

Formulation and in vitro evaluation of pentazocine transdermal delivery system.

The aim of this study was to prepare a pentazocine (PTZ) matrix-type transdermal drug delivery system (TDDS) using acrylic pressure-sensitive adhesives. Among the five Duro-Tak adhesive polymers tested (87-9301, 87-2677, 87-201A, 87-2196, 87-2852), in vitro dissolution studies demonstrated the highest PTZ release flux from the Duro-Tak 87-9301 matrix. In addition, the effects of permeation enhancers, isopropyl myristate (IPM) and glyceryl monocaprylate (GEFA-C(8)), and drug content on PTZ skin permeation from prepared patches were evaluated using Franz diffusion cells fitted with hairless mouse skin. IPM and GEFA-C(8) were found to produce effective flux of PTZ at a patch concentration of 10% w/w and 5% w/w, respectively. The PTZ flux increased linearly as the loading dose increased up to 30%, whereas no further increase in flux was observed at loading doses of 40% and 50% due to drug crystallization in the matrix. Thus, the highest skin permeation rate (24.2 microg/cm(2)/h) was achieved when 30% of PTZ was loaded in Duro-Tak 87-9301 with 10% IPM and 5% GEFA-C(8). These results demonstrate the feasibility of a novel narcotic-antagonist analgesic matrix-type TDDS for PTZ.

Effect of permeation enhancers on the in vitro percutaneous absorption of pentazocine.

The effect of permeation enhancers on the percutaneous absorption of pentazocine (PTZ) was investigated in excised hairless mice using Franz diffusion cells in vitro. The enhancing effect on the percutaneous absorption of PTZ from the isopropyl myristate (IPM) solution system was improved with glyceryl monocaprylate (GEFA-C(8)), which is a kind of glycerol ester of fatty acid (GEFA). The flux of PTZ through the skin was ca. 4 times higher compared with IPM alone, while a less enhancing effect of glyceryl dicaprylate (GEFA-DiC(8)) and glyceryl tricaprylate (GEFA-TriC(8)) on the skin permeation of PTZ was found. Moreover, maximum enhancement of PTZ flux was observed with glyceryl monocaproate (GEFA-C(6)) among various alkyl chains (C(2)-C(18)) of monoglycerides. These results indicated that the IPM solution system combination with GEFA may be used to develop a transdermal formulation with improved skin permeation of PTZ.

Compensatory effect by sigma1 (sigma1) receptor stimulation during alcohol withdrawal in mice performing an object recognition task.

Chronic alcohol consumption (CAC) provokes intense neurobiological alterations, which lead, notably, to an important abstinence syndrome upon withdrawal with deleterious cognitive consequences. We here examined the effect of activation or inactivation of the sigma(1) receptor during CAC withdrawal on the cognitive abilities of Swiss mice. Animals consumed an alcohol 10%/sucrose 30 g/l solution during 4 months. Control groups consumed only the sucrose vehicle solution. Then, animals experienced a progressive, 16 days long, CAC withdrawal, during which they were administered once daily with saline, igmesine (10 mg/kg i.p.), a sigma(1) receptor agonist, or BD1047 (10 mg/kg i.p.), a sigma(1) antagonist. Mice were then tested using an object exploration task, to evaluate their locomotor and exploratory activities and reactions to object habituation, spatial change or novel object presentation. CAC-treated animals showed augmentation of locomotion, anxiety and object exploration, which impeded correct reaction to object habituation, spatial change or novelty. Treatment with the sigma(1) ligands, ineffective in control groups, resulted in decrease of the hyper-responsiveness and restored habituation. However, correct reactions to spatial change and novelty were only produced by the sigma(1) agonist treatment. Moreover, the sigma(1) receptor hippocampal expression was increased in CAC-treated mice. Treatments with both sigma(1) ligands regulated its expression, but subcellular fractionation experiments revealed that the agonist treatment increased [(3)H](+)-pentazocine binding to sigma(1) sites in the plasma membrane fraction, while the antagonist maintained it only in the microsomal, putatively endoplasmic reticulum, fraction. In conclusion, CAC increased the sigma(1) receptor expression in the hippocampus of mice. Regulation of its expression during withdrawal, notably using a selective agonist, allowed not only to attenuate the CAC-induced hyper-responsiveness, but also to restore correct cognitive abilities.

Application of a generic physiologically based pharmacokinetic model to the estimation of xenobiotic levels in rat plasma.

The routine assessment of xenobiotic in vivo kinetic behavior is currently dependent upon data obtained through animal experimentation, although in vitro surrogates for determining key absorption, distribution, metabolism, and elimination properties are available. Here we present a unique, generic, physiologically based pharmacokinetic (PBPK) model and demonstrate its application to the estimation of rat plasma pharmacokinetics, following intravenous dosing, from in vitro data alone. The model was parameterized through an optimization process, using a training set of in vivo data taken from the literature and validated using a separate test set of in vivo discovery compound data. On average, the vertical divergence of the predicted plasma concentrations from the observed data, on a semilog concentration-time plot, was approximately 0.5 log unit. Around 70% of all the predicted values of a standardized measure of area under the concentration-time curve (AUC) were within 3-fold of the observed values, as were over 90% of the training set t1/2 predictions and 60% of those for the test set; however, there was a tendency to overpredict t1/2 for the test set compounds. The capability of the model to rank compounds according to a given criterion was also assessed: of the 25% of the test set compounds ranked by the model as having the largest values for AUC, 61% were correctly identified. These validation results lead us to conclude that the generic PBPK model is potentially a powerful and cost-effective tool for predicting the mammalian pharmacokinetics of a wide range of organic compounds, from readily available in vitro inputs only.

Age-related changes of the binding of [3h]SA4503 to sigma1 receptors in the rat brain.

We have recently developed 1-([3-O-methyl-11C]3,4-dimethoxyphenethyl)-4-(3-phenylpropyl) piperazine ([11C]SA4503) as a selective radioligand for mapping sigma1 receptors in the brain by positron emission tomography (PET). In the present short communication we evaluated the age-related changes of the binding of this ligand to sigma1 receptors in Fisher-344 rats (1.5-, 6-, 12-, and 24-month-old) by the in vitro binding assay. We also measured the binding of [3H](+)-pentazocine to sigma1 receptors and the binding of [3H]1,3-di-O-tolylguanidine to sigma2 receptors, which are current standard methods. The specific binding of the three radioligands increased age-dependently. Both Kd and Bmax values of the 24-month-old rats for each radioligand were significantly higher than those of the young rats (1.5- and 6-month-old). The increased numbers of both sigma1 and sigma2 receptor subtypes in the aged rats compensate for the lowered affinity, and rather enhanced the radioligand-receptor binding. The results contrast strikingly with the age-dependent decrease in the dopaminergic, cholinergic and glutamatergic receptors that are reported to be correlated with the sigma receptors, and indicate that a PET study with [11C]SA4503 to evaluate the aging process in humans would be of great interest.

Investigation on the influx transport mechanism of pentazocine at the blood-brain barrier in rats using the carotid injection technique.

The influx transport mechanism of pentazocine (PTZ) at the blood-brain barrier (BBB) was investigated in rats using the carotid injection technique. The uptake kinetics of PTZ into the rat brain exhibited saturability, which occurred by both nonsaturable and carrier-mediated transport processes. The in vivo kinetic parameters were estimated as follows: the maximal uptake rate (Jmax), 3.6 +/- 1.2 micromol/min/g brain and the apparent Michaelis constant (K1), 3.7 +/- 1.7 mM for the saturable component of PTZ into the brain, and the nonsaturable uptake rate constant (Kd), 0.06 +/- 0.04 ml/min/g brain. The uptake of PTZ by the brain was strongly inhibited by lidocaine, imipramine and propranolol, and also by H1-antagonists such as mepyramine, diphenhydramine. In addition, narcotic-antagonist analgesic (buprenorphine, butorphanol or eptazocine) and an opioid antagonist (naloxone) significantly inhibited PTZ transport. These results suggest that PTZ permeates into the brain via a carrier-mediated transport system, which may widely recognize the cationic drugs.

Investigation of transport mechanism of pentazocine across the blood-brain barrier using the in situ rat brain perfusion technique.

To characterize pentazocine (PTZ) transport across the blood-brain barrier (BBB), the cerebrovascular permeability-surface area product (PS(inf)) of PTZ was determined by a well-established in situ rat brain perfusion technique. The uptake kinetics of PTZ by the rat brain exhibited saturability, which indicates the simultaneous mechanisms of carrier-mediated transport and passive diffusion. The kinetic parameters were estimated as follows: maximal influx rate (V(max)), 27.2 +/- 5.2 nmol/s/g brain; apparent Michaelis constant (K(m)) for the saturable component of PTZ uptake, 2.9 +/- 0.5 mM; nonsaturable uptake rate constant (K(d)), 1.5 +/- 0.3 microL/s/g brain. BBB transport of PTZ was significantly inhibited by cationic drugs such as diphenhydramine, propranolol, and eptazocine (a narcotic-antagonist analgesic), but not by choline, suggesting that the PTZ transport system is shared by cationic drugs. Furthermore, co-perfusion of verapamil caused a significant (two-fold) increase in the BBB permeability to PTZ. This finding indicates that PTZ may be a substrate of the endogenous BBB efflux transport system, P-glycoprotein. These findings demonstrate that the primary mechanism governing the uptake of PTZ by the brain is carrier-mediated transport, not passive diffusion.

Multiple pathways of sigma(1) receptor ligand uptakes into primary cultured neuronal cells.

Although many antipsychotics have affinities for sigma receptors, the transportation pathway of exogenous sigma(1) receptor ligands to intracellular type-1 sigma receptors are not fully understood. In this study, sigma(1) receptor ligand uptakes were studied using primary cultured neuronal cells. [(3)H](+)-pentazocine and [(3)H](R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), used as a selective sigma(1) receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. sigma(1) receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na(+)-independent. Kinetic analysis of [(3)H](+)-pentazocine and [(3)H]MS-377 uptake showed K(m) values (microM) of 0.27 and 0.32, and V(max) values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [(3)H](+)-pentazocine was inhibited in the range 0.4-7.1 microM by all the sigma(1) receptor ligands used, including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective sigma(1) receptor ligand. In contrast, the inhibition of [(3)H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC(50), approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC(50), 4.5 microM). Moreover, kinetic analysis revealed that NE-100 inhibited [(3)H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [(3)H]MS-377. These findings suggest that there are at least two uptake pathways for sigma(1) receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In addition, pretreatment of cells with a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), a myosin light chain kinase inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)homopiperazine (ML-9), or microsomal Ca(2+)-ATPase inhibitors resulted in a reduction of the amount of sigma receptor ligand uptake. These findings suggest that the Ca(2+) pump on the endoplasmic reticulum and/or calmodulin-related events might be involved in the regulation of the uptake of sigma receptor ligands into primary neuronal cells.

Chitosan based pentazocine microspheres for intranasal systemic delivery: development and biopharmaceutical evaluation.

Bioadhesive chitosan microspheres (Ms) of pentazocine (Pz) for intranasal systemic delivery were prepared with the aim of avoiding the first pass effect, and thus improving the bioavailability and achieving sustained and controlled blood level profiles, as an alternative therapy to injection and to obtain improved therapeutic efficacy in the treatment of chronic pain such as cancer, trauma and post-operative pain, etc. The formulation variables were drug loading, polymer concentration, stirring rate during crosslinking and oils. The microspheres (Ms) were subjected to evaluation for physical characteristics, such as particle size, incorporation efficiency, swelling ability, in vitro bioadhesion, in vitro drug release characteristics and in vivo performance in rabbits. Application of in vitro data to various kinetic equations indicated matrix diffusion controlled drug delivery from chitosan Ms. Drug loading, polymer concentration and stirring speed influenced the drug release profiles significantly while oils had negligible effect. In vivo studies indicated significantly improved bioavailability of Pz from Ms with sustained and controlled blood level profiles as compared to i.v., oral and nasal administration of drug solution. Good correlation was observed between in vitro and in vivo data.

Targeting sigma receptor-binding benzamides as in vivo diagnostic and therapeutic agents for human prostate tumors.

Sigma receptors are known to be expressed in a variety of human tumor cells, including breast, neural, and melanoma tumors. A very high density (1.0-1.5 million receptors/cell) of sigma receptors was also reported in a human androgen-dependent prostate tumor cell line (LNCaP). In this study, we show that a very high density of sigma receptors is also expressed in an androgen-independent human prostate tumor cell line (DU-145). Pharmacological binding studies using the sigma-1-selective ligand [3H](+)-pentazocine showed a high-affinity binding (Kd = 5.80 nM, Bmax = 1800 fmol/mg protein). Similarly, binding studies with [3H]1,3-di-o-tolylguanidine in the presence of dextrallorphan also showed a high-affinity binding (Kd = 15.71 nM, Bmax = 1930 fmol/mg protein). Radioiodinated benzamide N-[2-(1'-piperidinyl)ethyl]-3-[125I]iodo-4-methoxybenzamide ([125I]PIMBA) was also shown to bind DU-145 cells in a dose-dependent manner. Three different radioiodinated benzamides, [125I]PIMBA, 4-[125I]iodo-N-[2-(1'-piperidinyl)ethyl]benzamide, and 2-[125I]-N-(N-benzylpiperidin-4-yl)-2-iodobenzamide, were screened for their potential to image human prostate tumors in nude mice bearing human prostate cells (DU-145) xenografts. All three compounds showed a fast clearance from the blood pool and a high uptake and retention in the tumor. Therapeutic potential of nonradioactive PIMBA was studied using in vitro colonogenic assays. A dose-dependent inhibition of cell colony formation was found in two different human prostate cells. These results demonstrate the potential use of sigma receptor binding ligands in non-invasive diagnostic imaging of prostate cancer and its treatment.

Quinolinic acid and sigma receptor ligand: effect on pyramidal neurons of the CA1 sector of dorsal hippocampus following peripheral administration in rats.

Male Wistar rats, weighing 200-220 g, were used in the study. Quinolinic acid and racemic pentazocine were administered alone or together. Quinolinic acid was administered intraperitoneally (i.p.) in a dose of 60 mmol, racemic pentazocine intramuscularly in a dose of 2 mg/kg, once every 24 h for 8 days. The control group received 1 ml of saline i.p. once daily for 8 days. Pentazocine alone produced no signs of alteration in the hippocampal formation. Quinolinic acid produced neurotoxic effect in the CA1 area of the hippocampal formation. The presence of the dark-degenerated pyramidal cells was a common sign of a delayed excitotoxic effect. Pentazocine added to quinolinic acid markedly attenuated the neurotoxic effect of quinolinic acid. In such cases, only single dark degenerated cells were seen.

Pharmacokinetics of pentazocine and its occupancy of opioid receptors in rat brain.

In order to assess quantitatively the pharmacodynamic process of pentazocine (PTZ), time courses of its plasma concentration and of the occupation of specific opioid receptors in the brain were investigated after intravenous (i.v.) administration of PTZ to rats. The plasma concentration of PTZ was determined by HPLC and the pharmacokinetic parameters were analyzed using nonlinear least-squares analysis. Measurement of ex vivo receptor occupation was made by comparing the specific [3H]naloxone (opioid receptor antagonist) binding in vitro to the crude P2-synaptosomal fractions between vehicle-treated rats (control) and PTZ-treated rats. Following the i.v. administration of PTZ, the occupancy of specific opioid receptors decreased rapidly until 10 min, depending on the two pharmacological doses (2.5 and 10 mg/kg). The results strongly suggest the fast binding kinetics of PTZ in terms of its association with and dissociation from specific opioid receptor sites in the brain in addition to its fast rate of disappearance from the brain compartment. Furthermore, we demonstrated that the time profile of receptor occupancy correlated well (r = 0.8650) with that of the unbound concentration in plasma until 120 min after the i.v. administration of PTZ to rats.

Distribution of drugs in various tissues in a brain dead man.

We examined the distribution of drugs in a 49-year-old brain-dead man. Our objective was to determine the possibility of diagnosing how and at what point the patient became brain dead. The presence of mepivacaine, pentazocine, lidocaine and thiamylal in various tissues, including seven regions of the brain were confirmed, using gas chromatography/mass spectrometry. Tissue-to-blood concentration ratios of mepivacaine, pentazocine and lidocaine in the brain were higher than these ratios in other tissues, while ratios of thiamylal were lower. Therefore, cerebral blood flow was likely to have ceased between the administration of the former drugs and that of the latter drug, in agreement with clinical records. Among seven regions of the brain, the ratios of the former three drugs were high in occipital and parietal lobes, and were low in the cerebellum and medulla oblongata. On the other hand, the ratios of the latter drug were high in the cerebellum and the medulla oblongata. Therefore, cerebral blood flow presumably ceased first in occipital and parietal lobes, and last in the cerebellum and the medulla oblongata. Based on these results, assessment of concentrations of drugs in human tissues, including various regions of brain is useful to determine the time and progression of brain death.

Dissociation of the motor effects of (+)-pentazocine from binding to sigma 1 sites.

Radioligand binding and behavioral studies were conducted to determine whether a relationship existed between the motor effects produced by (+)-pentazocine and its binding to sigma sites. Scatchard analyses revealed decreased [3H](+)-pentazocine binding in middle aged rats (5-6 months old) compared to young adult rats (2-3 months old). However, there was no difference between the extent of circling behavior or dystonia produced by microinjection of (+)-pentazocine into the substantia nigra or red nucleus in the older animals compared to the young adult rats. There was also a significant decrease in [3H](+)-pentazocine binding in rats chronically treated with haloperidol. Again, however, despite the reduction in [3H](+)-pentazocine binding, there was no difference between the extent of dystonia produced by unilateral intrarubral microinjection of (+)-pentazocine into animals chronically treated with haloperidol vs. saline. The postural changes produced by (+)-pentazocine could not be attenuated with coadministration of the putative sigma receptor antagonist BD1047 (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino) ethylamine), or the opiate receptor antagonist naloxone. However, the (+)-opiate, (+)-nordihydrocodeinone, partially attenuated the postural effects of (+)-pentazocine, despite its very low affinity for sigma 1, sigma 2, or opiate receptors. Taken together with previous studies, the results suggest that [3H](+)-pentazocine is a potent and selective probe for sigma 1 binding sites, but the in vivo effects of (+)-pentazocine cannot be fully attributed to actions through these sites. Some of the in vivo effects of (+)-pentazocine appear to involve other binding sites that are not detected under the conditions normally used in in vitro assays.